Rapid detection of pecan root-knot nematode, Meloidogyne partityla, in laboratory and field conditions using loop-mediated isothermal amplification

Abstract

Meloidogyne partityla is the dominant root-knot nematode (RKN) species parasitizing pecan in Georgia. This species is known to cause a reduction in root growth and a decline in the yields of mature pecan trees. Rapid and accurate diagnosis of this RKN is required to control this nematode disease and reduce losses in pecan production. In this study, a loop-mediated isothermal amplification (LAMP) method was developed for simple, rapid, and on-site detection of M. partityla in infested plant roots and validated to detect the nematode in laboratory and field conditions. Specific primers were designed based on the sequence distinction of the internal transcribed spacer (ITS)-18S/5.8S ribosomal RNA gene between M. partityla and other Meloidogyne spp. The LAMP detection technique could detect the presence of M. partityla genomic DNA at a concentration as low as 1 pg, and no cross reactivity was found with DNA from other major RKN species such as M. javanica, M. incognita and M. arenaria, and M. hapla. We also conducted a traditional morphology-based diagnostic assay and conventional polymerase chain reaction (PCR) assay to determine which of these techniques was less time consuming, more sensitive, and convenient to use in the field. The LAMP assay provided more rapid results, amplifying the target nematode species in less than 60 min at 70˚C, with results 100 times more sensitive than conventional PCR (~2-3 hrs). Morphology-based, traditional diagnosis was highly time-consuming (2 days) and more laborious than conventional PCR and LAMP assays. These features greatly simplified the operating procedure and made the assay a powerful tool for rapid, on-site detection of pecan RKN, M. partityla. The developed LAMP assay will facilitate accurate pecan nematode diagnosis in the field and contribute to the management of the pathogen.

Development and Validation of a Loop-Mediated Isothermal Amplification Diagnostic Method to Detect the Quarantine Potato Pale Cyst Nematode, Globodera pallida

“…For detection of different species of root-knot nematodes, several LAMP assays have been developed, such as for M. arenaria, M. hapla, Meloidogyne incognita, M. javanica [36], M. enterolobii [37], M. hapla [38], M. mali [39], M. chitwoodi and M. fallax [40]. Recently, a LAMP-based diagnostic assay was published for the pecan root-knot nematode, M. partityla [41]. In a variant assay to detect M. hapla, DNA from the root galls was directly crushed onto Flinders Technology Associates (FTA) cellulose cards and stored at room temperature for years and directly used as a template in LAMP reactions [30,38].…”

Differences in distribution and community structure of plant-parasitic nematodes in pecan orchards between two ecoregions of Georgia

“…are widespread in pecan orchards located in 23 different Georgia counties (4 and 19 counties from both Piedmont and Coastal Plain ecoregions, respectively). However, the RKN species is unknown in Georgia as we have only confirmed the presence of M. partityla in three counties (Jefferson, Houston, and Tift) located in Coastal Plain ecoregion of Georgia (Waliullah et al, 2020;Jagdale unpublished data). Nyczepir and Woods (2008) reported a wide distribution of M. partityla in Georgia, but they did not provide any specific distribution details.…”

Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry

“…The newly synthesized LAMP primer sets including F3, B3, FIP [F1c-F2], BIP [B1c-B2], LF and LB designed by Primer Explorer version 5 (Figure 4, Supplementary Table S1) using the RNA polymerase sigma-70 factor rpoD locus from Xfm sequence (Figure 1, Supplementary Table S1) were used to optimize the temperature conditions. Based on the previous studies and this study, the optimum 10× formulation of the LAMP primer mix was determined to be: 0.2 µM each of primers F3 and B3, 0.8 µM each of primers LF and LB, and 1.6 µM each of primers FIP and BIP [45,46]. To rule out the optimum reaction temperature, a gradient LAMP was performed from 66 to 73 • C using the 10× formulation of the six LAMP primers with 0.1 ng/µL of diluted Xfm pure culture DNA.…”

“…The aim of this study was to develop and validate simple methods to distinguish between Xff and Xfm isolates efficiently and rapidly. Unlike other methods for subspecies differentiation, the CAPS marker technique does not require the use of multiple PCR primers or subsequent sequencing [43,44], and LAMP is a unique approach widely used for plant pathogen diagnosis for its simplicity, specificity, and sensitivity [42,45,46]. In this study, we were able to identify and differentiate between Xff and Xfm isolates from blueberry using these two separate assays.…”